1. Field of the Invention
The present invention in the field of biochemistry and medicine is directed to angiogenesis-inhibitory polypeptides comprising a part of human kininogen, particularly, the D3 domain (HK-D3) and variants thereof, and their use in diagnosis and therapy of diseases associated with endothelial cell migration and proliferation. In particular these polypeptides are useful in treating subjects with cancer.
2. Description of the Background Art
Angiogenesis, the formation of new capillaries form pre-existing ones (Folkman, J., N. Engl. J. Med., 1971, 285:1182–1186; Hanahan D. et al., Cell, 1996, 86:353–364), is a normal part of embryonic development, wound healing and female reproductive function. However, angiogenesis also plays a pathogenic role in the establishment and progression of certain diseases. Cancer, rheumatoid arthritis and diabetic retinopathy are examples of such diseases (Carmeliet P. et al., Nature, 2000, 407:249–257). Anti-angiogenic therapy holds promise in inhibiting the progression of these diseases.
Angiogenesis can be triggered by several pro-angiogenic cytokines. In the setting of cancer, tumor cells under hypoxic conditions secrete vascular endothelial growth factor (VEGF) and/or fibroblast growth factor (bFGF). These proteins diffuse and bind to specific receptors on endothelial cells (ECs) in the local vasculature, perturbing the balance of pro- and anti-angiogenic forces in favor of angiogenesis. As a consequence of binding these proteins, ECs are activated to (a) secrete enzymes that induce remodeling of the associated tissue matrix, and (b) change the patterns and levels of expression of adhesion molecules such as integrins. Following matrix degradation, ECs proliferate and migrate toward the hypoxic tumor, resulting in the generation and maturation of new blood vessels.
Interestingly, many anti-angiogenic factors result from the degradation of matrix proteins—i.e., are a result of the action of pro-angiogenic enzymes. Examples include endostatin, a fragment of collagen XIII (O'Reilly, M. S. et al., Cell 1997, 88:277–285); kringle 5 of plasminogen (O'Reilly, M. S. et al., Cell, 994, 79:315–328) and PEX, the C-terminus non-catalytic subunit of MMP-2 (Brooks PC et al., Cell, 1998, 92:391–400).
The concept has emerged that, due to the abundance of pro-angiogenic factors, these anti-angiogenic molecules are unable to overcome the pro-angiogenic balance in a primary tumor. However, since they are secreted into circulation, these anti-angiogenic molecules are capable of inhibiting angiogenesis at other locations where tumor cells may have begun to invade. Consequently, micro-metastases comprising these tumor cells at these new locations remain dormant. This hypothesis explains the puzzling observation made by surgeons many years ago: at various times after surgical removal of a primary tumor in a patient with no obvious metastatic disease, the patient returns with advanced metastatic disease.
Thus, clinical intervention by treatment with one or more of the anti-angiogenic factors could inhibit the angiogenic process and halt tumor growth as well as metastasis. Significant evidence in the literature (cited above) supports this notion.
Biochemistry of High Molecular Weight Kininogen
Two forms of kininogen, high molecular weight kininogen (HK, Mr=120 kDa), and low molecular weight kininogen (LK, Mr=68 kDa), have been identified in human plasma (Jacobsen S. et al., Br J Pharm 29:25–36, 1967). HK is an α-globulin with a plasma concentration of 90 μg/ml (Proud D et al., J Lab Clin Med 95:563–5574, 1980) (FIG. 1), and LK is a α-globulin with a plasma concentration of 220 μg/ml (Muller-Esterl W et al., Biochim Biophys Acta 106:145–152, 1982). These proteins are derived from the alternative splicing of a single gene (Kitamura N et al., J Biol Chem 260:8610–8617, 1985), and share a common heavy (H) chain, which contains domains 1, 2 and 3, termed D1, D2 and D3 (Colman R W et al., Blood 90:3819–3843, 1997). However, while LK contains only a 4 kDa light (L) chain (D4L), the ˜46 kDa L chain of HK contains domains 5 and 6 (D5 and D6, respectively).
Each domain of HK has a unique function. For example, D1 binds calcium, and D2 inhibits calpain (Colman et al., supra). The cell binding regions of HK are contained within D3 and D5, while D6 binds plasma prekallikrein and coagulation Factor XI. In intact HK, D4 links the H and L chains; D4 also includes the nonapeptide, bradykinin (BK) which is released from HK by kallikrein via cleavage between Lys362-Arg363 and Arg371-Ser372, leaving behind a cleaved molecule consisting of a 62 kDa H chain and 56–62 kDa L chain, which are bonded by an intrachain disulfide between Cys10 and Cys596. A subsequent cleavage at a site near the N-terminus of D5, results in reduction of the Mr of the L chain to ˜45 kDa (Kaplan A P et al., Blood 70:1–15, 1987).
Released BK is a potent vasodilator and an agonist for ECs. Kallikrein-mediated cleavage of HK occurs on the EC surface, and may be mediated (a) directly by plasma kallikrein or (b) after binding of prekallikrein to cell-bound HK, followed by its activation to kallikrein by an EC cysteine protease. Thus the EC is an important site for HKa generation. Phorbol myristoyl acetate (PMA)-stimulated ECs bind increased amounts of HK (Colman et al., supra) suggesting acceleration of this process on “activated” ECs. The observation that ECs produce HK mRNA and protein further supports the physiological importance of this process (Schmaier A H et al., J Biol Chem 263:16327–16333, 1988).
The release of BK from HK is accompanied by a structural rearrangement in the remaining two-chain kininogen molecule, HKa and the acquisition of several novel properties. For example, cleavage of HK to HKa allows the latter to bind to artificial anionic surfaces (Colman et al., supra); interactions that are mediated by residues of the His-Gly-rich region within D5 of HKa (amino acids 420–458) (DeLacadena R A et al., Protein Sci 1:151–160, 1992; Kunapuli S P et al., J Biol Chem 268:2486–2492, 1993).
Furthermore, HKa, but not HK, is anti-adhesive, inhibiting the spreading of osteosarcoma and melanoma cells on vitronectin, and of ECs, platelets and mononuclear cells on vitronectin and fibrinogen (Asakura S et al., J Cell Biol 116:465–476, 1992). The structural rearrangement of HKa involves a change in the orientation of HKa domains relative to each other.
HK exists as a linear array of three linked globular regions, with the two peripheral regions connected by a thin strand (Colman R W et al., J Clin Invest 100:1481–1487, 1997). The strand may represent the disulfide bridge between D1 and D6, as it is no longer apparent following reduction. Studies with epitope-specific monoclonal antibodies (mAbs) determined that the globular domains on the ends of HK represent the prekallikrein-binding region (within D6 of the L chain) and the cysteine protease inhibitor region (D2 and D3 of the H chain), while the central nodule represents the anionic surface binding region within D5.
After kallikrein-mediated cleavage, the two-chain molecule, HKa, retains the trinodular structure, though the three globular regions rearrange in a pattern resembling vertices of a triangle. In this structure, the anionic surface binding and prekallikrein binding regions are more closely apposed. Because the EC binding regions within HK have been mapped to sites within D3 of the H chain and D5 of the L chain ((Reddigari S R et al., Blood 81:1306–1311, 1993; Herwald H et al., J Biol Chem 270:14634–14642, 1995; Hasan A et al., J Biol Chem 269:31822–31830, 1994; Hasan A et al., J Mol Biol 219:717–725, 1995) and since the latter regions in the linear sequence overlap extensively with the anionic surface binding regions of HKa, the orientation of the cellular binding regions within HK and HKa must differ. This conclusion implies that HK and HKa are likely to interact differently with ECs, a hypothesis supported by functional studies demonstrating that HKa, but not HK, is a potent inhibitor of proliferation and inducer of apoptosis in ECs.
Interactions of HK with ECs
A. Identification of Cell Binding Regions within HK
HK was reported to bind with high affinity to human umbilical vein ECs (HUVEC) (Reddigari et al., supra; van Iwaarden F et al., J Biol Chem 263:4698–4703, 1988; Zini J M et al., Blood 81:2936–2946, 1993; Hasan A et al., Blood 85:3134–3143, 1995). The presence of Zn2+ is an absolute requirement for binding, whereas Ca2+ either inhibited or had no effect on binding. Internalization of HK has also been reported (van Iwaarden F et al., Blood 71:1268–1276, 1988).
The binding of HK to ECs is mediated through interactions involving both its H and L chains, and several studies have led to the identification of specific regions that mediate binding within D3 (Herwald H et al., supra) and D5 (Hasan et al., J. Mol. Biol., supra) (one of which overlaps with the BK sequence within D4). These regions were identified by the ability of synthetic peptides with corresponding sequences to compete with intact, labeled HK for binding to HUVEC.
In contrast to HK, little information is available concerning the binding of HKa to ECs. In one study, cleavage of biotinylated HK by increasing amounts of kallikrein led to a progressive diminution in binding of the cleaved ligand. In contrast, others reported that HKa was more potent than unlabeled HK in inhibiting the binding of radiolabeled HK to ECs (IC50=73 nM for HKa vs 335 nM for HK) (Reddigari et al., supra). Although these IC50 values are difficult to reconcile with a reported Kd (30–40 nM) for the binding of HK to ECs, they nevertheless suggest differences between HK and HKa in their interactions with cells.
B. Endothelial Cell HK/HKa Receptors
HKa inhibition of EC proliferation in vitro is a unique property of HKa as HK, which binds to ECs, nevertheless lacks this antiproliferative effect. Moreover, the observed difference in binding to ECs exhibited by HK and HKa suggests potential differences in function. HKa could inhibit EC proliferation by several mechanisms. First, it might induce detachment of ECs from their matrix through direct interactions with integrins, thereby leading to interruption of integrin-mediated signaling and MAP kinase phosphorylation, leading to apoptosis. However, other than one report that single-chain HK binds to Mac-1 (αMβ2 or CD11b/CD18) on monocytes, there is no evidence for interactions of kininogen with integrins.
The binding of HKa to ECs was also not inhibited by a blocking antibody against the β3 integrin chain, suggesting that HKa does not interact with αvβ3, an integrin which plays an important role in angiogenesis (Colman R W et al, J Clin Invest 100:1481–1487, 1997). HKa might interact in either a specific or non-specific manner with an ECM protein(s), thereby preventing its interaction with an EC integrin receptor. However, there is no data to support this hypothesis. The fact that HKa inhibited the proliferation of HUVEC plated on fibronectin, gelatin, and Matrigel, suggested effects independent of matrix identity. HKa might inhibit the binding of growth factors to cellular glycosaminoglycans, such as heparan sulfate, or to specific growth factor receptors. However, this explanation is unlikely, since withdrawal of growth factors does not lead to EC apoptosis within 6 hours—a time frame in which HKa induced apoptotic changes.
McCrae's group recently observed that the cleaved form of HKa inhibited bFGF-stimulated angiogenesis in vivo. (Zhang J-C et al., FASEB J. 14:2589–600, 2000). In vitro, HKa potently inhibited the proliferation of HUVEC and human dermal microvascular ECs (HDMVEC), inducing EC apoptosis. Several peptides were identified with sequences corresponding to the binding regions within D3 and D5 of HKa that inhibited EC proliferation at low μM to nM concentrations. Comparison of the sequences of overlapping peptides used in these studies led to the identification peptides of 4–8 amino acids that mediated this activity. Compared to the antiproliferative effects, the anti-adhesive effects of HKa appear to be of less importance since EC adhesion was only modestly inhibited at HKa concentrations >100 nM, whereas anti-proliferative effects were observed at concentrations as low as ˜1 nM. McCrae (WO 00/35407; PCT/US99/28465) has described variants of the 8-mer peptide X1-Asn-Asn-Ala-Thr-Phe-Tyr-Phe-Lys-X2, which are discussed in the context of EXAMPLE I.